Fatemeh Faghihi, Jafar Amani, Milad Gholami, Iman Salahshourifar,
Volume 29, Issue 1 (3-2026)
Abstract
Autism Spectrum Disorder (ASD) and CDKL5 deficiency disorder are among the nervous and developmental disorder with genetic origin that requires accurate molecular models to study the mechanisms of disease and develop new treatments. The CRISPR / Cas9 genome editing system is an efficient tool to generate targeted changes in the genome. The aim of this study was to design and construct CRISPR / Cas9 vector carrier sgRNA for CDKL5 gene and to confirm the cloning and identity of the cell line used by molecular methods .
Materials and methods: in this experimental study, at first, specific primers were designed for region in the cdkl5 gene .Oligo nouclotide forward and reverse annealing and cloned in PX458 vector. After transformation of E. coli strains DH5, positive colonies were confirmed by PCR and sequencing methods. Also to ensure the identity of the HEK293T cell line, QF - PCR was used to determine the sex and chromosome study.
Results: colony - PCR results showed the expected band of 230 bp in selected colonies. Sequencing also confirmed insert sgRNA in the vector. The results of QF-PCR demonstrated specific peaks patterns for sex chromosomes that confirmed cell line identity.
Conclusion: the CRISPR / Cas9 target gene expression was successfully performed and molecular approvals were obtained for cloning and analysis of the cell line. This vector can be used for further studies of gene expression in cellular models.
